A complete theory describing the nucleation, aggregation, and subsequent crystallization of solvated molecules or ionic species does not currently exist, and a principal reason for this is the paucity of experimental evidence to support or refute theoretical hypotheses. Currently, a strong consensus in the art exists for a two step nucleation process. These steps are posited to comprise (1) the formation of clusters, solvated, but with some degree of chemical interaction and a degree of order beyond that found in the “normal” solvated state; and (2) the subsequent arrangement of the solvated species to a type of protocrystal. The latter step is believed to be the rate-determining step for crystallization.
One of the more promising methods of analysis currently being used to study crystal growth is atomic force microscopy. However, the information gained from the use of this technique is restricted to the understanding of epitaxial growth on existing crystal surfaces. Therefore, this method cannot be applied to the study of nucleation prior to the existence of a single unit cell.
With the successful demonstration of our dynamic chemical imaging in general, and dynamic Raman imaging in particular, new possibilities emerge for the molecular specific imaging of important time dependent phenomena in many varied fields, such as biology, organic chemistry, inorganic chemistry, biochemicals, and fabrication of semiconductor materials, to name a few. Raman scattering is extremely sensitive to crystal structure and even to orientation in soft materials. In particular, we can see the nucleation and aggregation that heretofore had been hidden.
Through the development of our dynamic chemical imaging capabilities, chemical insight into nucleation (prior to crystallization) and aggregation through spectral imaging of dynamic processes is now available to us for development through “Streaming Imaging” of crystal dissolution and subsequent recrystallization. This Streaming Imaging, or chemical imaging of dynamic processes, is now a reality and there is great potential to reveal many chemical and physical processes that have been “invisible” because of the absence of techniques for “seeing” transient processes.
Understanding and controlling crystallization is essential for the manufacture of products as varied as electronic devices, large-tonnage commodity materials, and high-value specialty chemicals such as pharmaceuticals. Yet understanding of the crystallization process remains limited, especially for organic, polymeric, and protein crystals. Once a crystal has formed, its internal structure can be determined by x-ray diffraction, but unraveling the key steps leading up to and during the process of crystallization requires tools that allow for control and microscopic visualization of crystal growth, particularly at the early stages that often determine crystal properties such as defect density, purity, size, morphology, and polymorphism (the ability of a material to adopt different crystal structures). The ability to view crystallization events directly, at the level of the individual growth unit, promises insights into the influence of experimental condition on crystallization at the near-molecular level, rather than by inference from characterization of bulk crystals.
In the area of biology, the occasional conversion of proteins from their intricately folded functional forms into thread-like molecular aggregates is not well understood. These transformations into an alternative form of protein structure are of much more than academic interest since such aggregates are linked to some of the most feared diseases of the modern era. These molecular aggregates are usually known as amyloids, or amyloid-like fibrils, and are perhaps most notorious for their association with Alzheimer's disease. However, amyloids are also involved in some twenty other protein “misfolding” disorders, including type II diabetes, the transmissible forms of the diseases epitomized by scrapie, “mad cow” disease in domesticated animals, and by kuru and Creutzfeldt-Jakob disease in humans. The proteins involved in these conditions are known as prions (proteinaceous infectious particles). Prions are increasingly turning up in different organisms, particularly yeast and other fungi. The yeast prions are not functionally or structurally related to their mammalian namesakes, and their ability to convert into fibrillar aggregates is coupled not just to disease but also to the inheritance of genetic traits. Proteins in amyloid fibrils are folded to produce a core region consisting of a continuous array of beta-sheets. Such sheets are a familiar type of protein motif, and here are made up of beta-strands that are oriented perpendicular to the fibril axis in an arrangement called a cross-beta structure. The ability to form this type of structure may be a generic feature of polypeptide chains, although the specific amino-acid sequence of the chain affects both the propensity to form fibrils and the way a given molecule is arranged within the fibrils. Knowledge of this latter aspect is vital for understanding the properties of protein forms such as prions, but has been seriously limited by the intractability of amyloid fibrils to the traditional methods of structural biology. Although much theoretical work has been published on the subject, there has never been much supporting experimental work because the right technological tools have not been available.
Additionally, embodiments of the disclosed method and apparatus may be used for visualizing, and therefore controlling, the existence of different crystalline forms of chemical compounds. Many chemical compounds can exist in multiple discrete crystalline forms. For example, graphite and diamond are discrete crystalline forms of elemental carbon. The property of being able to assume multiple crystalline forms is commonly designated polymorphism, and the different crystalline forms of the same compound are designated polymorphic forms or, more simply, polymorphs. Polymorphs of a single compound generally have chemical properties that vary in at least subtle ways. For instance, polymorphs can exhibit differences in melting points, electrical conductivities, patterns of radiation absorption, x-ray diffraction patterns, crystal shapes, dissolution rates, and solubilities, even though the polymorphs are made up of the same chemical.
In the context of pharmaceutically active compounds, differences among polymorphs can affect the pharmacological properties of the compound in significant ways. By way of example, the dissolution rate of a drug can greatly influence the rate and extent of bioavailability of the drug when administered by a selected route. Furthermore, the shelf stability of a drug compound can vary significantly, depending on the polymorphic form the drug assumes. In the U.S. and elsewhere, regulatory approval of a drug formulation often requires knowledge and description of the polymorphic form(s) of the drug that occur in the composition submitted for approval. This is so because approvability of a drug substance requires reproducibility in manufacture, dosing, and pharmacokinetic behavior of the drug. In the absence of such reproducibility, safety and efficacy of the drug cannot be sufficiently assured.
The polymorphic form(s) of a compound that are present in a composition is important in other industries as well. By way of example, the properties of dyes and of explosives can be strongly influenced by polymorphism. The crystalline form(s) present in a food product can affect the taste, mouth feel, and other properties of the product.
The crystal shape that a chemical compound assumes can be heavily influenced by the polymorphic form assumed by the compound. In turn, the bulk properties of a preparation of a compound in crystalline form(s) depend on the polymorphic form(s) assumed by the compound in the preparation. For instance, the flow characteristics, tensile strength, compressibility, and density of a powdered form of a compound will be determined by the polymorphs present in the preparation.
Various techniques are known for investigation of polymorphic forms of a compound that occur in the solid state. Such methods include polarized light microscopy (including hot-stage microscopy), infrared spectrophotometry, single-crystal X-ray and X-ray powder diffraction, thermal analysis, and dilatometry. In many instances, these methods can be limited by resolution of the method, polymorphic non-homogeneity of the analyte, similarity among polymorphs of the property analyzed, or other practical difficulties. In particular, compositions that contain multiple polymorphic forms of a compound can be difficult or impossible to analyze using such techniques.
Improved methods and apparatus for assessing the polymorphic forms of a compound, particularly in a solid particulate form and methods for influencing the polymorphic form assumed by a compound could overcome or limit the shortcomings identified above. Additionally, improved methods and apparatus are needed for visualizing the change of an attribute of a sample, such as, but not limited to, nucleation, aggregation, and subsequent crystallization of solvated molecules or ionic species, molecular specific imaging of time dependent phenomena, understanding and controlling crystallization, and conversion of proteins into prions. Obtaining a streaming image and/or comparison of spectra from a sample undergoing a change is necessary to realize the above goals.
Therefore, it is an object of the present disclosure to provide a method and apparatus for producing a streaming chemical image of photons scattered by, or emitted by, a sample where an attribute of the sample changes as a function of time.
It is another object of the present disclosure to provide a method and apparatus for determining a change in an attribute of a sample by detecting, analyzing, and comparing spectra of the sample where the attribute changes as a function of time.